Water soluble, randomly substituted partial N-partial O-acetylated chitosan, preserving compositions containing chitosan, and processes for making thereof

ABSTRACT

The present invention is directed to a water soluble, randomly substituted partial N-, partial O-acetylated chitosans or chitosan derivatives and methods of preparing water soluble, randomly substituted partial N-, partial O-acetylated chitosans or chitosan derivatives comprising the steps of dissolving the chitosan or chitosan derivative into an aqueous acidic solution and reacting the chitosan or chitosan derivative with an acetylating agent in the presence of a phase transfer reagent. The present invention is further directed to a pharmaceutical preserving composition comprising: (a) at least one chitosan or chitosan derivative and (b) at least one buffer solution, as well as methods of preserving contact lens solutions and disinfecting contact lens using such composition.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a Continuation-In-Part of U.S. applicationSer. No. 09/838,528, filed Apr. 19, 2001, which is aContinuation-In-Part of U.S. application Ser. No. 09/611,160 filed Jul.6, 2000, which claims priority to U.S. Provisional Application Ser. Nos.60/199,406, filed Apr. 21, 2000, and No. 60/202,548, filed May 10, 2000,which are all herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to novel water soluble, randomlysubstituted partial N-, partial O-acetylated chitosan or derivativesthereof, and preserving compositions containing water soluble, randomlysubstituted partial N-, partial O-acetylated chitosan, chitosan orderivatives thereof and novel processes for making water-solublerandomly substituted partial N-, partial O-acetylated chitosan, chitosanor derivatives thereof.

BACKGROUND OF THE INVENTION

[0003] Ophthalmic products intended for repeated use after opening, thatis “multi-dose” products, must be preserved to minimize contaminationwith microorganisms during use. Preservatives that are used inophthalmic solutions are often irritating to the eye, and at worst, maydamage eye tissue after repeated use. Preservative problems may beworsened in contact lens solutions when a contact lens that has beenexposed to a preservative in a lens care solution acts as a reservoirthat concentrates the preservative in the eye.

[0004] In the United States, acceptably preserved pharmaceuticalproducts, including ophthalmic, nasal and otic preparations, mustachieve minimum performance standards when tested according to theprocedures of the United States Pharmacopoeia Preservative Efficacy Test(PET). According to the PET protocol, adequately preserved formulationsmust reduce 0 day challenge inocula and 14 day re-challenge inocula ofthe bacteria Staphylococcus aureus, Pseudomonas aeruginosa andEscherichia coli by at least 99.99% (3 logs) within 14 and 28 days afterthe challenge date. In the fungal challenge portion of the PET,preserved formulations must not allow any growth of Aspergillus nigerand Candida albicans within 14 and 28 days following the 0 daychallenge. To demonstrate preservative efficacy for contact lens careproducts, a modified PET procedure is required by the FDA wherein are-challenge of the test solutions is done on day 14 after the 14 dayorganism concentrations are determined.

[0005] Chitosan, the de-acetylation product of chitin, is a non-toxicbiopolymer with weak antimicrobial activity. Heretofore, the use ofchitosan to preserve pharmaceutical compositions has been hampered byits insolubility at pH above 6 and also because the antimicrobialactivity of Chitosan in acidic solutions, by itself, is too low to meetPET requirements. Chitosan's water solubility at near neutral pH can beimproved by derivatization with hydrophilic functional groups, such ascarboxymethyl or glycol substituents, or by selective N-acetylation ofcommercially available chitosans.

[0006] Considerable efforts have been made to extend the watersolubility of chitosan at neutral pH. In Sannan et al., Makromol Chem.177, 3589 (1976), it was reported that, by treatment of chitin withalkali under homogeneous conditions, chitin with about 50% deacetylationbecame water-soluble. However, long reaction time and large quantitiesof solvent are required in some stages, including neutralization of thereaction mixture and removal of the resulting salt. This laboriousprocess would be troublesome especially in large-scale production.

[0007] Kurita et al., Carbohydrate Polymers 16, 83 (1991), alsodiscloses preparing water-soluble chitosan with about 50% N-acetylationby acetylating a 90% deacetylated chitosan with a complex solventsystem, comprising aqueous acetic acid/methanol/pyridine. Kurita et al.describes that the resultant partially N-acetylated chitosan is watersoluble, if the degree of acetylation is controlled at 50% and theacetyl groups are distributed randomly. However, the huge excess ofpyridine solvent used by the Kurita method made this processimpractical. Furthermore, the reaction products have limited watersolubility at neutral pH because heterogeneous reaction conditions wereemployed that restrict uniform, random acetylation. Specifically,Kurita's chitosan reactant was not soluble in the reaction mixture, butinstead it was dispersed as a swollen gel which hindered completeavailability of reaction sites. In this case, the acetylation reactionwould be favored in those chain segments that were most exposed and freeto the reaction mixture, while other parts of the gel would becomparatively less acetylated due to steric interference from adjacentpolymer chain segments. When taken as a whole, the polymer chain is notuniformly random, but instead is comprised of blocks of higher and loweracetylation.

[0008] Kubota et al., Polymer Journal. 29, 123 (1997), reported to havea facile preparation of water-soluble N-acetylated chitosan. In thisreference, the chitosan is degraded by treatment with NaBO₃, and thedepolymerized product is then N-acetylated with acetic anhydride inaqueous acetic acid. Since both physical-chemical and biologicalproperties of chitosan are dependent upon the chemistry of the polymer,such as the random distribution of a definite amount of acetyl groupsand the molecular weight of the polymer, this process, which involvesdepolymerization, might alter the biological properties of chitosan.

SUMMARY OF INVENTION

[0009] The present invention is directed to a pharmaceutical preservingcomposition comprising: (a) at least one chitosan or chitosanderivative, and (b) at least one buffer solution.

[0010] The present invention is further directed to a method ofpreserving a contact lens solution, comprising mixing a contact lenssolution with the composition comprising: (a) at least one chitosan orchitosan derivative, and (b) at least one buffer.

[0011] Moreover, the present invention relates to a method ofdisinfecting a contact lens, comprising soaking the contact lens withthe composition comprising: (a) at least one chitosan or chitosanderivative, and (b) at least one buffer solution for a suitable periodof time.

[0012] The present invention also is directed to a compositioncomprising (a) at least one chitosan or chitosan derivative, and (b) atleast one buffer solution, wherein the at least one chitosan or chitosanderivative is prepared by a method comprising the steps of dissolvingthe at least one chitosan or chitosan derivative into an aqueous acidicsolution and reacting the chitosan with an acetylating agent in thepresence of a phase transfer reagent.

[0013] The present invention is further directed to a process forproducing a water soluble, randomly substituted partial N-, partialO-acetylated chitosan or chitosan derivative, comprising the steps ofdissolving a chitosan or chitosan derivative in an aqueous acidicsolution and reacting the chitosan or chitosan derivative with anacetylating agent in the presence of a phase transfer reagent. In afurther aspect, the invention relates to the product made by such aprocess.

[0014] The present invention is further directed to a water soluble,randomly substituted partial N-, partial O-acetylated chitosan orderivative thereof represented by the formula (I),

[0015] wherein R₁, R₂ and R₃ are independently H or C(O)CH₃, wherein thechitosan or derivative thereof is partially acetylated such that R₁ hasa degree of substitution of C(O)CH₃ of from about 24 to about 55%, andR₂ has a degree of substitution of C(O)CH₃ of from about 1 to about 60%,m is greater than 25, wherein the partial N-, partial O-acetylatedchitosan or derivative thereof is randomly substituted and is watersoluble.

[0016] In another aspect, the invention provides a pharmaceuticalpreserving composition comprising:

[0017] (a) at least one water soluble, randomly substituted partiallyN-, partial O-acetylated chitosan or derivative, of formula (I),

[0018] (b) and at least one buffer solution.

[0019] In yet another aspect, the invention provides a pharmaceuticalpreserving composition comprising the product formed from mixingcomponents (a) and (b) as described in the above aspect.

[0020] In another aspect, the invention provides a phamaceuticalpreserving composition comprising:

[0021] (a) at least one water soluble, randomly substituted partial N-,partial O-acetylated chitosan or derivative,

[0022] (b) and at least one buffer solution,

[0023] wherein the at least one water soluble, randomly substitutedpartial N-, partial O-acetylated chitosan or chitosan derivative isprepared by a method comprising the step of reacting at least onerandomly substituted partial N-, partial O-acetylated chitosan orchitosan derivative with a base in a solvent.

[0024] In another aspect, the invention provides a contact lens solutioncomprising the pharmaceutical preserving composition as described above.

[0025] In another aspect, the invention provides a contact lens solutioncomprising the product formed from mixing components (a) and (b) asdescribed above.

[0026] In another aspect, the invention provides a process for producinga water soluble, randomly substituted partial N-, partial O-acetylatedchitosan or chitosan derivative, comprising the step of reacting arandomly substituted partial N-, partial O-acetylated chitosan orchitosan derivative with a base in a solvent.

[0027] In another aspect, the invention provides a product produced bythe method of reacting a water soluble, randomly substituted partial N-,partial O-acetylated chitosan or chitosan derivative with a base in asolvent.

[0028] In other aspects, the invention provides for products made by theprocesses of the invention.

[0029] Additional advantages of the invention will be set forth in partin the description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

[0030] The present invention may be understood more readily by referenceto the following detailed description of preferred embodiments of theinvention and the Examples included therein.

[0031] Before the present compounds, compositions, articles, devices,and/or methods are disclosed and described, it is to be understood thatthis invention is not limited to specific synthetic methods, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

[0032] As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anacyl” includes mixtures of acyl groups, reference to “a halogen”includes mixtures of two or more such halogens, and the like.

[0033] Ranges may be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

[0034] In this specification and in the claims which follow, referencewill be made to a number of terms which shall be defined to have thefollowing meanings:

[0035] References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

[0036] A weight percent of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

[0037] “Aqueous acidic solution” means an aqueous solution having a pHbelow 7.0.

[0038] By the term “effective amount” of a compound or property asprovided herein is meant such amount as is capable of performing thefunction of the compound or property for which an effective amount isexpressed. The exact amount required will vary from process to process,depending on recognized variables such as the compounds employed and theprocessing conditions observed. Thus, it is not possible to specify anexact “effective amount.” However, an appropriate effective amount maybe determined by one of ordinary skill in the art using only routineexperimentation.

[0039] By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.

[0040] The term “water soluble” as used herein to describe the watersoluble chitosans of the present invention, is meant to includechitosans or derivatives thereof having a water solubility of at leastabout 0.2% where solubility is measured by the test described forExamples 8-28 of the specification. Using this method, the watersolubility of the randomly substituted partial N-, partial O-acetylatedchitosan of the present invention is in one aspect at least 0.2%, inother aspects is up to 2%, and in other aspects (if greater than 0.200 gof chitosan is used in the test) is higher than 2%. Water solubilitiesof the chitosans of the present invention may be even higher than 2% orless than 0.2% when measured by other test methods. In such cases, thewater solubility is dependent on the molecular weight of the polymer,the viscosity of the resulting aqueous chitosan solution and theconditions of the solubility test chosen.

[0041] By the term “randomly substituted” is meant a random substitutionof acetyl groups on the chitosan main chain, which contributes to thewater solubility or hydrophilicity of the resultant chitosan polymer.

[0042] By the term water soluble, “partial N-, partial O-acetylatedchitosan” or derivative thereof is meant a poly(N-,O-acetylated-D-glucosamine).

[0043] By the term “degree of deacetylation” is meant the percentage offree amino groups on the water soluble, chitosan or chitosan derivative.The percent of N-acetylation can be caluculated from the deacetylationvalue. The terms N-acetylation or O-acetylation are also referred to asthe degree of substitution with C(O)CH₃ on either N or O.

[0044] It is to be understood that greater than 50% N-acetylation issometimes described in the art as a chitin. However, the term “chitosan”is used throughout the invention herein to include chitosans and, if theN-acetylation is greater than 50%, to include chitins.

[0045] By the term “heterogeneous conditions” is meant that all or partof the reaction is carried out in a solid or highly swollen state, i.e.,gel.

[0046] By the term “homogenous conditions” is meant that the reaction iscarried out completely in a solution.

[0047] The present invention is a preserving composition forpharmaceutical products. The preserving composition can be used invarious ophthalmic products such as contact lens rinsing, lubricating,cleaning and storage solutions, artificial tear solutions and ophthalmicdrugs. The compositions of the instant invention may also be used topreserve otic and nasal solutions.

[0048] Contact lens solutions in particular present a specialpreservative challenge because lens wearers are usually exposed to thepreserving agents for many years on a daily basis. The possibility thatthe lens wearer can experience discomfort or develop sensitivity to thepreservative is even higher than would be the case in short-termexposure. Typical contact lens solution preserving agents used in theprior art are sorbic acid, thimerosal, or DYMED™ (polyaminopropylbiguanide).

[0049] The composition of this invention comprises at least one chitosanor chitosan derivative, and at least one buffer solution. Thecomposition of this invention additionally may contain at least onebiocidal adjuvant. Compositions of the present invention contain thesecomponents in amounts to be effective as pharmaceutical preservingcompositions useful for preserving pharmaceutical products, includingophthalmic, nasal and otic preparations.

[0050] One preferred embodiment is used as a contact lens solutionpreservative. Another preferred embodiment is used as a contact lensdisinfection regimen. When the composition comprised of at least onechitosan or chitosan derivative and at least one buffer solution is usedin a method to preserve a contact lens solution, the contact lenssolution is mixed with the composition. When the composition comprisedof at least one chitosan or chitosan derivative and at least one buffersolution is used in a contact lens disinfection regimen, the contactlens is rinsed and rubbed with the composition, and the contact lensthen soaks in the composition for a suitable period of time, such as notless than 15 minutes, more preferably for not less than 1 hour, evenmore preferably not less than four hours. Preferably, the soaking occursat room temperature; however, any suitable temperature may be employed.

[0051] In a preferred embodiment, the chitosan and chitosan derivativesof the present invention have the additional advantage of being capableof performing several functions normally requiring other ingredients.For instance, in a preferred embodiment, the chitosan or chitosanderivative may, in addition to its preserving role, act as a naturalsurfactant, and aid in lens cleaning by emulsifying lens proteins andlipids away from the lens surface into solution. Furthermore, chitosan,as a polymeric saccharide, can be used in a preferred embodiment as asolution thickening agent and lens lubricant thereby enhancing lenscomfort by reducing lens drying rate. As such, the chitosan or chitosanderivative in one embodiment of this invention has a demulcent effect soas to enhance lens wearer comfort.

[0052] Example chitosan or chitosan derivatives include chitosan salts,water-soluble chitosan, water-soluble, randomly substituted partial N-,partial O-acetylated chitosan, chitosan oligosaccharide, carboxymethylchitosan, and hydroxyalkyl chitosan. The hydroxyalkyl substituents ofthe hydroxyalkyl, chitosans and the carboxymethyl substituents of thecarboxymethyl chitosans could be attached to any of the pendant nitrogenor oxygen groups on the chitin or chitosan ring subunit. Specificpreferred hydroxyalkyl chitosans include but are not limited to,hydroxyethyl chitosan (also known as glycol chitosan), hydroxypropylchitosan, dihydroxypropyl chitosan, hydroxybutyl chitosan anddihydroxybutyl chitosan.

[0053] Example water soluble, randomly substituted partial N-, partialO-acetylated chitosan derivatives include such salt thereof,oligosaccharide thereof, carboxymethyl chitosan thereof, andhydroxyalkyl chitosan thereof. The hydroxyalkyl substituents of suchhydroxyalkyl chitosans and the carboxymethyl substituents of suchcarboxymethyl chitosans could be attached to any of the pendant nitrogenor oxygen groups on the chitin or chitosan ring subunit. Specificpreferred hydroxyalkyl chitosans of the partial N-, partial O-acetylatedchitosan, include but are not limited to, hydroxyethyl chitosan (alsoknown as glycol chitosan), hydroxypropyl chitosan, dihydroxypropylchitosan, hydroxybutyl chitosan and dihydroxybutyl chitosan.

[0054] In an embodiment, a water soluble, randomly substituted partialN-, partial O-acetylated chitosan or derivative thereof represented bythe following formula (I)

[0055] wherein R₁, R₂ and R₃ are independently H or C(O)CH₃, wherein thechito san or derivative thereof is partially acetylated such that R₁ hasa degree of substitution of C(O)CH₃ of from about 24 to about 55%, andR₂ has a degree of substitution of C(O)CH₃ of from about 1 to about 60%,

[0056] m is greater than 25,

[0057] wherein the partial N-, partial O-acetylated chitosan orderivative thereof is randomly substituted and is water soluble.

[0058] The term “m” is the number of repeat units in the water soluble,chitosan or polymer chain. In one aspect m is about 100,000, but inother aspects m can be higher. The molecular weight range of the watersoluble chitosan or polymer chain herein refers to the weight averagemolecular weight. The weight average molecular weight of the watersoluble chitosan or polymer is typically at least about 5,000. In oneaspect the weight average molecular weight can be up to about 3,000,000,but in other aspects can be higher.

[0059] It is a separately surprising finding of one embodiment of thisinvention that chitosan or chitosan derivatives with certain buffersolutions such as borate or phosphate buffers, have higher antimicrobialactivity as compared, for example, to similar formulations in citrate,and tromethamine (tris) buffers and in water. Thus, in one embodiment,the buffer solution may be comprised of a borate buffer. Suitable boratebuffers include, but are not limited to, boric acid, sodium borate,potassium tetraborate, potassium metaborate, and mixtures of the same.In another embodiment, the buffer solution may be comprised of aphosphate buffer. Suitable phosphate buffers include, but are notlimited to sodium dihydrogen phosphate and disodium hydrogen phosphate,and mixtures of the same.

[0060] The present invention includes a biocidal adjuvant. The biocidaladjuvant may be used against, for example, bacteria, fungi, and viruses.One advantage of the present invention is the surprising synergisticpreservative effect of the composition. Suitable biocidal adjuvantsinclude, but are not limited to, disodium ethylenediaminetetracetic acid(EDTA), nitrilotriacetic acid, andethyleneglyco-bis(β-amino-ethylether)-N,N-tetraacetic acid.

[0061] The present composition may contain several ingredients toperform the intended function of the composition. One possibleadditional component may be used to allow the composition to have anosmotic pressure near that of normal lachrymal fluids. Such a functionmay be achieved, for instance, by a tonicity agent, such as sodiumchloride, potassium chloride or glycerol.

[0062] One feature of a preferred contact lens solution embodiment ofthe present invention is that proteins are stabilized against denaturingas compared to commercial multi-purpose contact lens solutions. In oneembodiment, this effect may be accomplished by adding at least onesurfactant to the composition. The surfactant may also aid in thecleaning of the lens. Typical surfactants include, but are not limitedto, Pluronics® or poloxamers, which are block copolymers of ethyleneoxide and propylene oxide, or Tetronics® or poloxamine, which are blockcopolymers resulting from addition of ethylene oxide and propylene oxideto ethylene diamine. Other surfactants that may be used in the inventioninclude, but are not limited to, tyloxapol, octoxynols, nonoxynols, andTweens® or polyoxyethylene sorbitan fatty acid esters.

[0063] The contact lens solutions of the present invention may, inanother embodiment, contain viscosity agents to provide lubrication tothe eye. Typical viscosity agents include polymeric saccharides such asdextran, cellulose derivatives such as carboxymethyl cellulose andhydroxypropyl methylcellulose, polyvinyl alcohol,polyvinylpyrrolidinone, polyethylene glycol, and glycerin.

[0064] The present compositions have at least minimal preservingactivity. In one embodiment, the biocidal activity of the composition issufficient to meet the performance criteria of the Preservative EfficacyTest (“PET”) of the USP (United States Pharmacopoeia) as modified by theFDA. As such, the present compositions reduce 0 day challenge inoculaand 14 day re-challenge inocula of the bacteria Staphylococcus aureus(ATCC No. 6538), Pseudomonas aeruginosa (ATCC No. 9027) and Escherichiacoli (ATCC No. 8739) by at least 99.99% (3 logs) within 14 days afterthe challenge and re-challenge dates, each. In the fungal challengeportion of the PET, the present composition does not allow any growth ofAspergillus niger (ATCC No. 16404) and Candida albicans (ATCC No. 10231)within 14 days following a 0 day challenge and a 14 day re-challenge. Assuch, the present invention may be used in a method of preserving acontact lens solution, wherein the contact lens solution is mixed withthe composition.

[0065] In one embodiment, the composition of the present invention has anear neutral pH. This pH condition is preferred for compatibility withthe organism, such as the human eye. As such, one preferred pH of theinvention is from 6 to 8, preferably 6.6 to 7.8, and more preferably 6.8to 7.2. Insofar as the antimicrobial activity alone of the compositionis concerned, the lowest pH in the specified range is preferred. Givensuch preferred pH ranges, in one preferred embodiment, the chitosan orchitosan derivatives of the present invention are soluble atpharmaceutically acceptable pH levels. Another embodiment includeschitosan or chitosan derivatives that are near neutral soluble, meaningwater soluble, from pH 6 to 8.

[0066] The chitosan and chitosan derivatives described in the presentinvention may be prepared by any method recognized in the art.Alternatively, in one preferred method, which is a method of oneembodiment of the present invention, water-soluble, randomly substitutedpartial N-, partial O-acetylated chitosan and chitosan or chitosanderivative is prepared by dissolving the chitosan or chitosan derivativein an aqueous acidic solution and reacting the chitosan with anacetylating agent in the presence of at least one phase transferreagent. The preparation of the water soluble, randomly substitutedpartial N-, partial O-acetylated chitosan or chitosan derivative thereofis carried out in a homogenous solution, which provides for the randomacetylated substitution. The acetylating agent and phase transferreagent(s) employed are used in an effective amount to be suitable forpreparing the water-soluble, randomly substituted partial N-, partialO-acetylated chitosan and chitosan or chitosan derivative. In apreferred embodiment, the water soluble, randomly substituted partialN-, partial O-acetylated chitosan and chitosan preferably dissolves insolutions with near neutral pH values, such as from pH 6.0 to 8.0.Aqueous acidic solution refers to pH less than 7 and is typically theacidic pH used in the art for acetylation under heterogeneousconditions.

[0067] The acetylating agent acetylates the chitosan. As such, any knownacetylating agent may be used. Example acetylating agents include, butare not limited to, acetyl halides, and acetic anhydride. A preferredacetylating agent is acetic anhydride.

[0068] The phase transfer reagent may be comprised of any phase transferreagents known in the art. In general, the phase transfer reagent worksacross the water and organic phases. Suitable phase transfer reagentsinclude, but are not limited to, those described in “Phase-TransferCatalysis,” Starks, C., et.al. Chapman & Hall, 1994, which isincorporated by reference in its entirety. Example phase transferreagents include, but are not limited to, quaternary ammonium salts(Eq.I), quaternary phosphonium salts (Eq. II), crown ethers (Eq. IIIa-IIIc),and pyridinium salts (Eq. IV).

[A]w[B]x[C]y[D]zN+Q  (I)

or

[A]w[B]x[C]y[D]zP⁺Q⁻  (II)

where

[0069] each of w, x, y and z is an integer from 0 to 4 and w+x+y+z=4

[0070] Q is a counter-ion selected from F⁻, Cl⁻, Br⁻, I⁻, CH₃COO⁻, OH⁻,HSO₄ ⁻, NO₃ ⁻, PF₆ ⁻, BF₄ ⁻, HCOO⁻ and H₂PO₄ ⁻; and

[0071] A, B, C and D are each selected from C₁-C₁₈ alkyl, phenyl inwhich the phenyl ring is unsubstituted or substituted by C₁-C₈ alkyl,C₁-C₈ alkoxy, halo, hydroxy, phenoxy, nitro, carboxy, acetamido, oraryl, benzyl, cycloalkyl have 5-6 ring member or heterocyclic ringsystem.

[0072] In one preferred embodiment, quaternary ammonium salts (Eq. I)and quaternary phosphonium salts (II) include, but are not limited to,tetra C₁-C₄ alkyl ammonium halides, such as tetrabutylammonium bromide(“TBABr”), tetramethylammonium chloride (“TMACI”), tetrabutylammoniumdihydrogen phosphate (“TBADHP”), and tetrabutyl ammonium iodide(“TBAI”); benzyl tri C₁-C₄ alkylammonium halides, such asbenzyltriethylammonium chloride (“BTEACI”); and tetra C₁-C₁₈ phosphoniumhalides, such as tetrabutyl phosphonium bromide (“TBPBr”) andhexadecyltributyl phosphonium bromide (“HDTRPBr”).

[0073] A preferred embodiment includes a number of crown ethers (Eq.IIIa to IIIc) in practicing the present invention.

[0074] where X=O or S, independently selected for each X

[0075] /=1 to 3

[0076] In one preferred embodiment, suitable crown ethers according toEq. IIIa include, but are not limited to, 12-crown-4, 15-crown-5,18-crown-6 and 1,4,7,10,13,16-hexathiacyclooctadecane.

[0077] where m=1 to 3

[0078] In one preferred embodiment, suitable crown ethers in accordancewith Eq. IIIB include, but are not limited to, benzo-12-crown-4,benzo-15-crown-5 and benzo-18-crown-6.

[0079] where n=1 to 3

[0080] p=1 to 3

[0081] R₃=H, C₁ to C₄ alkyl or halogen

[0082] In one preferred embodiment, example crown ethers suitable forEq. IIIc include, but are not limited to, dicylohexano-18-crown-6,dicyclohexano-24-crown-8, dibenzo-18-crown-6, dibenzo-21-crown-7,dibenzo-24-crown-8, dibenzo-30-crown-10,di-tere-butyl-di-benzo-18-crown-6 and ′4-bromobenzo-18-crown-6.

[0083] Pyridinium salts (Eq. IV) may also be used in practicing thepresent invention.

[0084] where R₁=C₁ to C₁₈ alkyl, benzyl or carboxymethyl

[0085] R₂=C₁ to C₄ alkyl, chloro, fluoro, bromo, hydroxy, C₁ to C₄alkoxy or alkoxycarbonyl

[0086] X=counter ion of F, Cl, Br, I or p-toluene sulfonate.

[0087] Example pyridinium salts of Eq.IV include, but are not limitedto, C₁ to C₁₈ alkyl pyridinium halides, such as 1-dodecylpyridiniumchloride and 1-cetylpyridinium bromide, 1-benzyl pyridinium halides, and1-benzyl-3-hydroxypyridinium chloride.

[0088] In another embodiment, which is a method of one embodiment of thepresent invention, the water-soluble chitosan or chitosan derivative isprepared by a method comprising the step of reacting at least one watersoluble, randomly substituted partial N-, partial O-acetylated chitosanor chitosan derivative with a base in a solvent.

[0089] The base may be comprised of any bases known in the art. Examplebases include, but are not limited to, alkaline hydroxides, such aspotassium hydroxide or sodium hydroxide, and alkaline carbonates, suchas sodium carbonate, or trisodium phosphate.

[0090] The solvent may be comprised of any solvent known in the art.Example solvents include, but are not limited to, alcohols, such asmethanol, ethanol, or isopropanol, ethers such as diethyl ether or,tetrahydrofuran, polar solvents, such as dimethyformamide, dimethylsulfoxide or, N-methyl pyrrolidinone and ketones such as acetone or2-butanone.

[0091] This invention can be further illustrated by the followingexamples of various embodiments, although it should be understood thatthese examples are included merely for purposes of illustration and arenot intended to limit the scope of the invention unless otherwisespecifically indicated. The starting materials are commerciallyavailable unless otherwise described. All percentages are by weightunless otherwise described.

EXAMPLES

[0092] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds, compositions, articles, devices and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers (e.g., amounts, temperature,etc.), but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, percent is percent byweight, temperature is in ° C. or is at ambient temperature, andpressure is at or near atmospheric. Example 1: Isotonic aqueous contactlens solution containing glycol chitosan Glycol Chitosan 0.25% PluronicF68 ™ (BASF Corporation) 0.05% Ethylenediaminetetraacetic acid, 0.05%disodium salt dihydrate (EDTA) Borate Buffer: q.s. 100.00 mL SodiumBorate Decahydrate 0.08% Boric Acid 0.72% Ultrapure Water q.s. 100.00 mLSodium Hydroxide Solution (0.5 M) q.s.* pH = 6.9 Sodium Chloride q.s.Osmotic pressure = 300 mOsm

[0093] This solution was prepared by dissolving glycol chitosan,Pluronic F68™ and EDTA in approximately 90% of the required volume ofborate buffer. After all of the first components had dissolved,additional borate buffer was added to reach the desired volume.Sufficient volume of 0.5M sodium hydroxide solution was added to adjustthe pH to 6.9. Sodium chloride was then added to adjust the osmoticpressure. The solution was sterilized by filtering through a 0.45 micronfilter. The preserving efficacy of the solution was tested against thebacteria Staphylococcus aureus (ATCC No. 6538), Pseudomonas aeruginosa(ATCC No. 9027), Escherichia coli (ATCC No. 8739), and the fungiAspergillus niger (ATCC No. 16404) and Candida albicans (ATCC No. 10231)according to the modified USP preservative efficacy test (PET) proceduredescribed in the May, 1997 edition of Premarket Notification (510(k))Guidance Document for Contact Lens Care Products (Developed by the U.S.Department of Health and Human Services, Food and Drug Administration,Center for Devices and Radiologic Health).

[0094] Following this procedure, the test solution was initiallychallenged with at least 10⁵ microorganisms/mL (cfu/mL) for each speciestested, in duplicate, and at day 14, a re-challenge of the testsolutions was done wherein the viable concentration of each organismtype was adjusted to at least 10⁴ cfu/ml. The numbers of survivingmicroorganisms were determined at day 14, prior to the rechallengeinoculum adjustment, and at day 28. The test solution was deemed to beeffectively preserved if viable bacteria were reduced at least 3 logs ondays 14 and 28, and if viable fungi at days 14 and 28 were less than orequal to the challenge concentrations, (i.e. a log reduction of 0 ormore). As shown below the results of the PET indicate that Example 1 iseffectively preserved. TABLE 1-a Preservative efficacy test results forExample 1 Average Organism Log Reduction Microorganism 14 days 28 daysEffectiveness¹ Escherichia coli (Ec) 3.6 3.7 Pass Pseudomonas aeruginosa(Pa) 5.7 3.6 Pass Staphylococcus aureus (Sa) 5.1 3.3 Pass Candidaalbicans (Ca) 1.1 2 Pass Aspergillus niger (An) 1.5 1 Pass

[0095] Denaturing of tear proteins on soft contact lenses is a commonproblem. Once proteins denature on the lens, they are difficult toremove, reduce lens clarity, may cause allergic reactions for thewearer, and can act as attachment sites for infectious microorganisms.Lysozyme, especially is a potentially troublesome tear protein insofaras high water (˜55% water) contact lenses are concerned because lysozymeis a positively charged protein that is readily attracted to thenegatively charged lens surface. An in vitro assay was developed todetermine the ability of a test solution to retard lysozyme denaturing.In this assay, a 1% stock solution of lysozyme in isotonic boratebuffered saline (pH=7.0) is freshly prepared, and an aliquot of thisstock solution is mixed with an equal aliquot of a test solution. Theresulting mixture is heated at 75° C. for 15 minutes in a hot waterbath. After the mixture is removed from the heating bath, it is allowedto cool to room temperature before it is visually inspected for signs ofprotein denaturing as evidenced by the formation of a white precipitate.

[0096] Example #1 was evaluated using the described lysozyme assay incomparison to several commercial contact lens multi-purpose solutions,and the results from the assay represented in Table 1-b indicate thatonly Example #1 prevented lysozyme denaturing. TABLE 1-b Comparison ofprotein denaturation for Example 1 and commercial contact lensmulti-purpose solutions Appearance after Lysozyme Assay Test SolutionIngredients (75° C., 15 min.) COMPLETE ® Comfort Phosphate Buffer,Potassium & Sodium Precipitate Plus ™ (Allergan) Chloride, EdetateDisodium, Poloxamer 237, Hydroxypropyl methylcellulose, PHMB (1 ppm)ReNu MultiPlus ® (Bausch & Borate Buffer, Sodium Chloride, EdetatePrecipitate Lomb) Disodium, Poloxamine, Hydroxyalkylphosphonate, DYMED ™(1 ppm) Opti-Free ® Express ® (Alcon Citrate Buffer, Sodium Chloride,Edetate Precipitate Laboratories, Inc.) Disodium (0.05%), POLYQUAD ® (10ppm) Chlorhexidine Solution Borate Buffer, Sodium Chloride, PrecipitateChlorhexidine (50 ppm) Chlorhexidine Diacetate Borate Buffer, SodiumChloride, Precipitate Dihydrate Chlorhexidine diacetate dihydrate (50ppm) Borate Buffered Saline Borate Buffer, Sodium Chloride Hazy Solution(Control) Slight Precipitate Example 1 Borate Buffer, Sodium Chloride,Glycol Clear Solution Chitosan, Poloxamer 188, EDTA No Precipitate

[0097] Ocular irritation and in vitro biocompatibility was alsoevaluated for the Example 1 formulation. The degree of ocular irritationand epithelial cell layer staining was evaluated in 6 rabbits inaccordance with methods proposed by Draize J H, Woodard G, and Calvery HO: Methods for the Substances Applied Topically to the Skin and MucousMembranes. J. Pharmacol. Ext. Ther. (1944)82: 377-390. After apreliminary examination and Draize scoring of both eyes, each rabbitreceived eight (8) hourly 10 microliter instillations of Example 1 tothe surface of the right eye only, with the left untreated eye servingas a control. Within 1 hour after the last application of test solutionand again after 24, 48 and 72 hours, all eyes were evaluated inaccordance with the Draize scoring method. Slit-lamp biomicroscopicexamination using the McDonald-Shadduck scoring method (McDonald, T. O.and Shadduck J. A. 1977. Eye Irritation. Pages 162-166 in F. N. Marzulliand H. I. Maibach, eds. Advances in Modern Toxicology, Vol. 4,Dermatotoxicology and Pharmacology. Halsted Press, John Wiley & Sons,Inc., New York.) was also done at the end of the day of the solutioninstillations. The Draize and McDonald-Shadduck scoring for the test andcontrol eyes for all rabbits were “0”, meaning that Example 1 wasnon-irritating to the ocular surface of the rabbit eye.

[0098] The in vitro biocompatibility study of Example 1 was based on theagar diffusion test described in USP/NF 22 (87) Biological ReactivityTests In-Vitro. In this evaluation, a filter disc with a 0.1 ml aliquotof Example 1, and appropriate negative and positive control discs wereeach placed on duplicate agarose surfaces directly overlaying confluentmonolayers of L-929 mouse fibroblast cells. After incubating at 37 C. in5% CO₂ for 24-26 hours, the cultures were examined, revealing thatExample 1 showed no evidence of causing cell lysis or toxicity, thusmeeting the biocompatibility requirements of the USP (see table 1-c).TABLE 1-c In vitro biocompatibility evaluation results for Example 1Test/Control Sample Zone of Articles¹ No. Lysis (mm) Grade² Reactivity¹Example 1 1 0.0 0 None 2 0.0 0 None Filter Disc Control 1 0.0 0 None 20.0 0 None Positive Control 1 5.0 3 Moderate 2 5.0 3 Moderate NegativeControl 1 0.0 0 None 2 0.0 0 None

Example 2

[0099] This example illustrates the activity of different hydroxyalkylchitosan solutions against Escherichia coli in a 28 day USP PreservativeEfficacy Test (PET). Solutions 2a-e were prepared as described inExample 1, using the following recipe.

Example Formulations #2a-e

[0100] 0.05% EDTA

[0101] 1.00% Boric Acid

[0102] Ultrapure Water (q.s. adj 100.00 mL)

[0103] 0.5M Sodium Hydroxide(q.s. adj pH=6.9)

[0104] Sodium Chloride (q.s. adj mOsm=300)

[0105] a: control; b=0.25% glycol chitosan (SIGMA Chemical); c=0.25%hydroxypropyl chitosan (Austin Chemical Co.); d=0.25% hydroxybutylchitosan (Austin Chemical Co.); e=0.25% di-hydroxypropyl chitosan(Technology Resource International Corporation).

[0106] The conditions of the PET were the same as those for Example 1except that a re-challenge inoculum was not introduced at day 14. Forthis test, E. coli only was chosen as the screening microorganismbecause earlier tests showed that it was typically more resistant thanother PET bacteria to chitosan antimicrobial formulations. Thus,antimicrobial activity against E. coli was deemed predictive of efficacyagainst the other PET microorganisms.

[0107] Referring to Table 2, it can be seen that all of the testsolutions met the requirements of the USP PET for E. coli, namely thatthe number of viable bacteria were reduced by at least 3 logs by day 14following the initial bacterial challenge, and the concentrations of thetest bacteria decreased from the 14 day levels during the remainder ofthe 28 day test period. TABLE 2 Preservative efficacy of solutions 2a-2eagainst Escherichia coli Average E. coli Log Reduction Formulation 14days 28 days Effectiveness¹ 2a (control) 0.9 2.5 Fail 2b (glycolchitosan) 4.2 4.7 Pass 2c (hydroxypropyl chitosan) 3.9 4.3 Pass 2d(hydroxybutyl chitosan) 4.3 4.8 Pass 2e (dihydroxypropyl chitosan) 4.25.3 Pass

Example 3

[0108] This example illustrates the effect of pH on the antimicrobialactivity of glycol chitosan. The test organism that was evaluated inExample 3 is Pseudomonas aeruginosa (ATCC No. 9027), a microorganismthat is a particular problem in a common contact lens associated eyeinfection, infectious keratitis.

Example 3 Formulations

[0109] Glycol chitosan (Sigma Chemical)  0.5% Pluronic ™ F68 (BASFCorporation) 0.05% EDTA 0.05% Sodium borate decahydrate 0.08% Boric acid0.72% Ultrapure Water q.s. adj 100.00 mL Sodium hydroxide solution (0.5M) q.s. pH = 6.6, 7.2 or 7.8 Sodium chloride q.s. mOsm = 300 ± 10

[0110] TABLE 3 Comparison of the antimicrobial activity of Example 3against Pseudomonas aeruginosa Cfu Pseudomonas aeruginosa after 24hours^(1,2) PH = 6.6 pH = 7.2 pH = 7.8 2 184 >1000

[0111] The table above shows the average number of surviving colonies onthe 10⁵ recovery plates that were prepared 24 hours after challengingthe test formulations with 106 cfu/mL Pseudomonas aeruginosa. As can beseen from this data, the pH 6.6 and 7.2 formulations of glycol chito sanwere more effective in killing P. aeruginosa in 24 hours than the glycolchitosan formulation at pH=7.8.

Example 4

[0112] This example illustrates the antimicrobial activity of awater-soluble, randomly substituted partial N-, partial O-acetylatedchitosan formulation wherein the randomly substituted, water-solublepartial N-, partial O-acetylated chitosan was prepared according to themethod disclosed in Example 10.

[0113] Example 4 was formulated as follows: 500 ppm of the watersoluble, randomly substituted partial N-, partial O-acetylated chitosanwas dissolved in borate buffer (from Example 1) and 250 ppm EDTA wasadded. 0.5M sodium hydroxide solution was used to adjust the pH of thesolution to 7.0, the osmotic pressure of the solution was adjusted withsodium chloride to 300 mOsm, and the solution was sterile filteredthrough a 0.45 micron membrane.

[0114] The antimicrobial activity was determined for Example 4 at days14 and 28 according to the methods of the FDA modified USP preservativeefficacy test as described in Example 1. The results summarized in Table4 show that Example 4 passed the requirements of the preservativeefficacy test. TABLE 4 Preservative efficacy test results for Example 4Average Organism Log Reduction After 14 and 28 days MicroorganismEffectiveness¹ Day 14 Day 28 Escherichia coli (Ec) 4.8 3.8 PassPseudomonas aeruginosa 4.4 4.2 Pass (Pa) Staphylococcus aureus (Sa) 3.73.0 Pass Candida albicans (Ca) 1.2 0.8 Pass Aspergillus niger (An) 0.90.9 Pass

Example 5

[0115] Example 5 illustrates the effect of various buffers on theantimicrobial activity of a water-soluble, randomly substituted partialN-, partial O-acetylated chitosan formulation. The water-soluble,randomly substituted partial N-, partial O-acetylated chitosan inExample 5 was prepared according to the method disclosed in Example 10.

Example 5

[0116] Isotonic aqueous contact lens solution containing water-soluble,randomly substituted partial N-, partial O-acetylated chitosan inborate, phosphate, tris and citrate buffers. Concentration RandomlySubstituted, water-soluble partial N-, partial O-acetylated chitosan0.10% according to Example 10. Ethylendiaminetetraacetic acid, disodium0.05% salt dihydrate (EDTA) Buffer (borate, phosphate, tris orcitrate)*: q.s.** 100.00 mL Sodium hydroxide solution (0.5 M) q.s.** pH= 6.9 Sodium chloride q.s.** Osmotic pressure = 300 mOsm

[0117] The four solutions listed above were prepared as described forExample 4. The antimicrobial activity against E. coli was determined foreach solution at days 14 and 28 using the preservative efficacy testmethods described in Example 1. The results of the antimicrobialactivity test data in Table 5 reveal that the antimicrobial activity ofthe borate buffered solution was more than 2 logs higher on days 14 and28 than that of the other solutions, and the activity of the phosphatebuffered randomly substituted partial N-, partial O-acetylated chitosanwas higher than the TRIS and citrate buffered randomly substitutedpartial N-, partial O-acetylated chitosans. TABLE 5 Comparison of theantimicrobial activity against E. coli of water-soluble, randomlysubstituted partial N-, partial O-acetylated chitosan in borate,phosphate, tris and citrate buffers. Average Log Reduction E. coliExample 5, Buffer Type Day 14 Day 28 Borate 5.2 5.7 Phosphate 2.4 2.9TRIS 1.5 2.0 Citrate 2.0 0.9

Example 6

[0118] Example 6 illustrates the importance of EDTA in combination withrandomly substituted partial N-, partial O-acetylated chitosan toachieve preservative efficacy against Escherichia coli.

[0119] The composition of solutions 6a-h is shown below. Randomlysubstituted water- soluble partial N-, partial O- Poloxamer EDTA Ex. No.acetylated chitosan (ppm) 188 (ppm) (ppm) 6-a 1000  0 500 6-b   0  0 5006-c 1000  0  0 6-d   0  0  0 6-e 1000 500 500 6-f   0 500 500 6-g 1000500  0 6-h   0 500  0

[0120] The water-soluble, randomly substituted partial N-, partialO-acetylated chitosan used in the above test solutions was preparedaccording to the method described in Example 10. The above-listedingredients are dissolved in borate buffer as in Example 1. In additionthe pH of each solution was adjusted to 7.0 with 0.5M sodium hydroxidesolution and the osmolality of each solution was adjusted to 300mOsmoles with sodium chloride.

[0121] The antimicrobial activity of the solutions 6a-h against E. coliwas determined at days 14 and 28, according to the methods of the PET asdescribed in Example 1, and the 10 results are summarized in Table 6.TABLE 6 Antimicrobial activity against E. coli for Examples 6a-h AverageLog Reduction E. coli Preservative Example No. Day 14 Day 28Effectiveness¹ 6-a 4.0 4.9 Pass 6-b 1.9 1.7 Fail 6-c 2.1 0.9 Fail 6-d0.6 0.6 Fail 6-e 5.2 5.1 Pass 6-f 1.9 1.9 Fail 6-g 1.9 1.5 Fail 6-h 0.60.5 Fail

[0122] As can be seen in table 6, only solutions 6-a and 6-e caused theminimum 3 log reduction on days 14 and 28 that is required todemonstrate preservative efficacy against E. coli. By comparison, theantimicrobial activity of the corresponding control solutions 6-b (for6-a) and 6-f (for 6-e) in which randomly substituted partial N-, partialO-acetylated chitosan was removed, and the control solutions 6-c (for6-a) and 6-g (for 6-e) in which EDTA was removed, was less than halfthat of the randomly substituted partial N-, partial O-acetylatedchitosan/EDTA solutions. Thus, it appears from this data, that EDTA andrandomly substituted partial N-, partial O-acetylated chitosan areacting synergistically to provide the unexpected result that higherantibacterial activity against E. coli is obtained with randomlysubstituted partial N-, partial O-acetylated chitosan and EDTA than witheither ingredient formulated without the other. TABLE 7 Preservativeefficacy test results for chitosan oligosaccharide solutions AverageOrganism Log Reduction after 14 and 28 days E. coli P. aeruginosa S.aureus C. albicans A. niger Passed Test Solution 14 d 28 d 14 d 28 d 14d 28 d 14 d 28 d 14 d 28 d PET? Oligo. Chitosan in Water¹ 0.1 +0.4 +0.3+1.2 4.9 4.9 0.3 0.2 0.2 0.3 No Oligo. Chitosan in Water + 1.9 0.9 +0.20.0 3.1 2.6 0.4 0.3 0.1 +0.1 No EDTA^(1,2) Oligo. Chitosan in Borate 5.84.8 5.9 4.0 4.4 4.2 4.3 3.7 1.3 0.2 Yes Buffer + EDTA^(1,2,3)

Example 7

[0123] In this example the preservative effectiveness of solutions ofchitosan oligosaccharide are evaluated wherein the comparative solutionsinclude (1) water, (2) water with EDTA, and (3) borate buffer with EDTA.The pH and osmolality of the test solutions were adjusted to 7.0 and 300mOsmoles, respectively, as described in Example 1, and the preservativetest conditions are described in Example 1. It can be seen from the datashown in Table 7, that neither of the water solutions of chitosanoligosaccharide provided the 3 log reduction of P. aeruginosa and E.coli at day 14 and day 28 that is required to pass the modified USPpreservative effectiveness test with re-challenge at day 14. Incontrast, the oligosaccharide formulated in borate buffer waseffectively preserved since it reduced the concentrations of all of thebacteria tested by more than 3 logs, and prevented the growth of thefungi, C. albicans and A. niger.

[0124] Solubility Test

[0125] In the following examples 8-28 and comparative samples A-C, amixture of 0.200 g of a sample of chitosan in 10 ml of DI water wasstirred at room temperature for approximately eighteen hours. Thesolution was filtered through #1 qualitative filter paper, and thecontainer was washed with a small amount of deionized water. Thecombined filtrate was then placed in a weighed aluminum weighing dishand dried in an vacuum oven at around 60° C. The observed weightdifference is the weight of soluble solid. The results are shown inTables 8-11 as solubility in water (%), whereby 2% is the maximummeasurable solubility attainable under the conditions of this test(based on 0.200 g of chitosan in 10 ml of water). Some water solublechitosans of the present invention have an actual solubility greaterthan 2%. To determine if such actual solubility is greater than 2%, morethan 0.200 g of chitosan must be used (for 10 ml water). Some watersoluble, randomly substituted partial N, partial O-acetylated chitosansmay have higher water solubility than 2% when evaluated according to theconditions of other solubility methods.

[0126] Two samples were prepared according to Kurita's process; however,both samples have rather poor solubility in water at neutral pH value.

Comparative Example A

[0127] Following the procedure described in Kurita et al., CarbohydratePolymers 16, 83 (1991), method D, a solution of 3.0 g of chitosan with84% deacetylation in 80 ml of 10% aqueous acetic acid was diluted with80 ml of methanol and poured into 1000 ml pyridine to give a highlyswollen precipitate. 7.7 g of acetic anhydride was added at roomtemperature and after stirring for five hours, the mixture was pouredinto 3 liters of acetone. The precipitate was collected by filtration,washed with acetone, and dried to obtain 3.31 g of solid. The degree ofdeacetylation value and O-acetylation value were determined by the ¹HNMRmethod referenced in Example 8. (Although Kurita does not disclose NMRdata, our NMR data of the Kurita product indicated the presence of bothN-, and O-acetylation.)

Comparative Example B

[0128] Comparative example B was prepared by proceeding in a mannersimilar to that described in comparative example A, except 11.1 g ofacetic anhydride was used. There was obtained 3.45 g of solid. Thedegree of deacetylation value and O-acetylation value were determined bythe ¹HNMR method referenced in Example 8.

Comparative Example C

[0129] Comparative example C was prepared according to the proceduredescribed in example 4, except no tetrabutylammonium bromide was added.There was obtained 4.17 g of solid. The degree of deacetylation valueand O-acetylation value were determined by the ¹HNMR method referencedin Example 8.

Example 8

[0130] A viscous solution was prepared by dissolving 13.5 g of chitosanwith deacetylation degree of 84% in 600 ml of 10% acetic acid solution.1.35 g of benzyltriethylammonium chloride was added, followed by 38.5 gof acetic anhydride. The resulting mixture was stirred at roomtemperature for approximately eighteen hours. 400 ml of methanol wasadded and the mixture was stirred for an additional 30 minutes. Thereaction mixture was then transferred into an additional funnel followedby the slow addition of 2400 ml of acetone with good agitation. Theprecipitate was collected and then washed with acetone until nodetectable amount of acetic acid remained. The resultant solid weighed12.24 g. The degree of deacetylation (DD) value and O-acetylation valuewere determined by ¹H NMR method. (A. Hirai, H. Odani and A. Nakajima:Polymer Bulletin 26, 87 (1991)). DD refers to the percentage ofN-deacetylation. The percentage of N-acetylation (degree of substitutionwith C(O)CH₃) is 100-DD.

Example 9

[0131] In a procedure similar to that described in example 8, 10.25 g ofchitosan, 450 ml of 10% acetic acid, 2.56 g of tetrabutyl ammoniumbromide and 26.05 g of acetic anhydride were reacted at room temperaturefor approximately eighteen hours to get 11.19 g of solid. The degree ofdeacetylation (DD) value and O-acetylation value were determined by theIHNMR method referenced in example 8.

Example 10

[0132] Proceeding in a manner similar to that described in example 8,13.5 g of chitosan, 600 ml of 10% acetic acid, 3.375 g oftetrabutylammonium bromide and 17.2 g of acetic anhydride wereinteracted to get 14.72 g of solid. The degree of deacetylation (DD)value and O-acetylation value were determined by the ¹HNMR methodreferenced in example 8.

Example 11

[0133] Following the procedure described in example 8, 3.347 g ofchitosan, 150 ml of 10% acetic acid, 0.335 g of tetrabutyl ammoniumbromide and 5.725 g of acetic anhydride were interacted to get 3.14 g ofsolid. The degree of deacetylation (DD) value and 0-acetylation valuewere determined by the ¹HNMR method referenced in example 8.

Example 12

[0134] Example 12 was prepared by a procedure similar to that describedin example 11, except tetrabutylammonium bromide was replaced bybenzyltriethylammonium chloride. There was obtained 3.69 g of solid. Thedegree of deacetylation value and O-acetylation value were determined bythe ¹HNMR method referenced in example 8.

Example 13

[0135] Example 13 was prepared following the procedure described inexample 11, except 9.54 g of acetic anhydride was used to obtain 4.04 gof solid. The degree of deacetylation (DD) value and O-acetylation valuewere determined by the ¹HNMR method referenced in example 8.

Example 14

[0136] Example 14 was prepared following a procedure similar to thatdescribed in example 11, except tetrabutylammonium bromide was replacedby tetramethyl ammonium chloride. There was obtained 3.54 g of solid.The degree of deacetylation value and O-acetylation value weredetermined by the ¹HNMR method referenced in Example 8.

Example 15

[0137] Example 15 was prepared according to the procedure described inexample 11, except tetrabutylammonium bromide was replaced bytetrabutylammonium iodide. There was obtained 4.33 g of solid. Thedegree of deacetylation value and O-acetylation value were determined bythe ¹HNMR method referenced in example 8.

Example 16

[0138] Example 16 was prepared according to the procedure described inexample 11, except tetrabutylammonium bromide was replaced withtetrabutylammonium dihydrogen phosphate. There was obtained 4.13 g ofsolid. The degree of deacetylation value and O-acetylation value weredetermined by the ¹HNMR method referenced in Example 8. TABLE 8 Effectof quaternary ammonium salts on the water solubility of randomlysubstituted partial N-, partial O-acetylated chitosan DD valueSolubility (%) O-acetylation in water Samples Catalyst (by NMR) (%) (byNMR) (%) Vanson 84   0   0.025 chitosan Compare None 63.5 16.9 0*  sample A Compare None 66.7 12.3  0.10# sample B Compare None 55.6 21.3 0.015# sample C Example 8 BTEACl (1:4)** 54.4 19.4 1.87 Example 9 TBABr(1:4) 57.9 18.0 2.00 Example 10 TBABr (1:4) 74.1 17.3 1.83 Example 11TBABr (1:10) 64.3 16.8 1.93 Example 12 BTEACl (1:10) 67.2 23.7 2.00Example 13 TBABr (1:10) 58   32.5 1.95 Example 14 TMACl (1:10) 58.4 19.92.00 Example 15 TBAI (1:10) 57.7 46.0 2.00 Example 16 TBADHP (1:10) 59.848.8 1.89

Example 17

[0139] Example 17 was prepared by following the procedure described inexample 8, by 10 reacting 13.5 g of chitosan, 600 ml of 10% acetic acid,1.35 g of hexadecyltributyl phosphonium bromide and 38.5 g of aceticanhydride at room temperature for approximately eighteen hours to get13.61 g of solid. The degree of deacetylation value and O-acetylationvalue were determined by the ¹HNMR method referenced in example 8.

Example 18

[0140] Proceeding in a manner similar to that described in example 8,13.5 g of chitosan, 600 ml of 10% acetic acid, 1.35 g of tetrabutylphosphonium bromide and 38.5 g of acetic anhydride were interacted toget 13.32 g of solid. The degree of deacetylation value andO-acetylation value were determined by the ¹HNMR method referenced inexample 8. TABLE 9 Effect of quaternary phosphonium salts on the watersolubility of randomly substituted partial N-, partial O-acetylatedchitosan DD value Solubility (%) O-acetylation in water Samples Catalyst(by NMR) (%) (by NMR) (%) Vanson 84   0   0.025 chitosan Comparison None63.5 16.9 0*   sample A Comparison None 66.7 12.3  0.10# sample BComparison None 55.6 21.3  0.015# sample C Example 17 HDTBPBr 54.1 22.41.86 (1:10)** Example 18 TBPBr (1:10) 54.4 32.8 1.76

Example 19

[0141] A viscous solution was prepared by dissolving 10.0 g of chitosanwith deacetylation degree of 90% in 225 ml of 20% acetic acid solution.1.0 g of 18-crown-6 was added, followed by 28.6 g of acetic anhydride.The resulting mixture was stirred at room temperature for approximatelyeighteen hours. The reaction mixture was transferred into an additionfunnel and 1600 ml of acetone was added dropwise with good agitation.The precipitate was collected and washed with acetone until nodetectable amount of acetic acid was left. The resulting solid weighed11.84 g. The degree of deacetylation (DD) value and O-acetylation valuewere determined by the HNMR method referenced in Example 8.

Example 20

[0142] Proceeding in a manner similar to that described in example 19,10.0 g of chitosan, 225 ml of 20% acetic acid, 1.0 g ofcis-dicylohexano-18-crown-6 and 28.6 g of acetic anhydride were combinedto get 12.92 g of solid. The degree of deacetylation (DD) value andO-acetylation value were determined by the ¹HNMR method referenced inExample 8.

Example 21

[0143] Following the procedure described in example 19, 10.0 g ofchitosan, 225 ml of 20% acetic acid, 1.0 g of 15-crown-5 and 28.6 g ofacetic anhydride were combined to get 12.52 g of solid. The degree ofdeacetylation (DD) value and O-acetylation value were determined by the¹HNMR method referenced in Example 8.

Example 22

[0144] Proceeding in a manner similar to that described in example 19,10.0 g of chitosan, 225 ml of 20% acetic acid, 1.0 g ofdibenzy-18-crown-6 and 18.6 g of acetic anhydride were reacted at roomtemperature for approximately eighteen hours. After the reaction, themixture was quenched slowly into 1500 ml of isopropanol. The precipitatewas collected and washed with isopropanol until no detectable amount ofacetic acid remained. The resulting solid weighed 12.52 g. The degree ofdeacetylation (DD) value and O-acetylation value were determined by theIHNMR method referenced in Example 8. TABLE 10 Effect of crown ethers onthe water solubility of randomly substituted partial N-, partialO-acetylated chitosan DD value Solubility (%) O-acetylation in waterSamples Catalyst (by NMR) (%) (by NMR) (%) Vanson 84   0   0.025chitosan Compare None 63.5 16.9 0*   sample A Compare None 66.7 12.3 0.10# sample B Compare None 55.6 21.3  0.015# sample C Example 1918-crown-6 47.4 55.1 1.87 (1:10)** Example 20 DC-18-crown-6 48.6 41.32.0  (1:10) Example 21 15-crown-5 51.3 47.5 1.70 (1:10) Example 22DB-18-crown-6 53.6 30.6 1.79 (1:10)

Example 23

[0145] Following the procedure described in example 22, 10.0 g ofchitosan, 225 ml of 20% acetic acid, 1.0 g of cetylpyridinium bromidemonohydrate and 28.6 g of acetic anhydride were combined to get 11.56 gof solid. The degree of deacetylation (DD) value and O-acetylation valuewere determined by the ¹HNMR method referenced in Example 8.

Example 24

[0146] Proceeding in a manner similar to that described in example 19,10.0 g of chitosan, 225 ml of 20% acetic acid, 1.0 g of1-dodecylpyridinium chloride monohydrate and 28.6 g of acetic anhydridewere combined to get 12.992 g of solid. The degree of deacetylation (DD)value and O-acetylation value were determined by the ¹HNMR methodreferenced in Example 8.

Example 25

[0147] Following the procedure described in example 19, 10.0 g ofchitosan, 225 ml of 20% acetic acid, 1.0 g of 1-benzyl-3-hydroxypyridinium chloride and 28.6 g of acetic anhydride were interacted toget 11.43 g of solid. The degree of deacetylation (DD) value andO-acetylation value were determined by the ¹HNMR method referenced inexample 8. TABLE 11 Effect of pyridinium salts on the water solubilityof randomly substituted partial N-, partial O-acetylated chitosan DDvalue Solubility (%) O-acetylation in water Samples Catalyst (by NMR)(%) (by NMR) (%) Vanson 84   0   0.025 chitosan Compare None 63.5 16.90*  sample A Compare None 66.7 12.3  0.10# sample B Compare None 55.621.3   0.015# sample C Example 23 CPB (1:10)** 50.9 22.6 2.0 Example 24DPCl (1:10) 48.1 57.5 2.0 Example 25 BHPCl (1:10) 50.4 30.8 2.0

Example 26

[0148] In a procedure similar to that described in Example 8, 4.5 g ofchitosan, 400 ml of 10% acetic acid, 1.0 g of tetrabutylammonium bromideand 9.0 ml of acetic anhydride were reacted at room temperature forapproximately eighteen hours to get 5.6 g of solid. The degree ofdeacetylation (DD) value was 75.9% and O-acetylation value was 12.3%determined by ¹H NMR method referenced in Example 8. The solubility inwater was 1.86%.

Example 27

[0149] A mixture of 1.5 g of O-acetylated chitosan described in Example26, 1.0 g of potassium hydroxide and 200 ml of methanol was stirred atroom temperature for 18 hours. The resulting product was filtered andwashed with 2×100 ml of isopropyl alcohol. The dried solid weighed 1.12g. The degree of deacetylation (DD), value was 76.2% and O-acetylationvalue was 1.2% determined by the IHNMR method referenced in Example 8.The solubility in water was 2.0%.

Example 28

[0150] Example 28 was prepared by following the procedure described inExample 8, by reacting 5.0 g of chitosan, with a deacetylation degree of86%, 405 ml of 5% acetic acid, 0.34 g of tetrabutylammonium bromide and8.4 ml of acetic anhydride to get 5.63 g of solid. The degree ofdeacetylation value (DD) was 64.7% and the O-acetylation value was 2.5%determined by the ¹HNMR method referenced in Example 8. The solubilityin water was 2.0%.

[0151] Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

[0152] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A water soluble, randomly substituted partial N-,partial O-acetylated chitosan or derivative thereof represented by theformula I,

wherein R₁, R₂ and R₃ are independently H or C(O)CH₃, wherein thechitosan or derivative thereof is partially acetylated such that R₁ hasa degree of substitution of C(O)CH₃ of from about 24 to about 55% and R₂has a degree of substitution of C(O)CH₃ of from about 1 to about 60%,and m is greater than 25, wherein the partial N-, partial O-acetylatedchitosan or derivative thereof is randomly substituted and is watersoluble.
 2. A pharmaceutical preserving composition comprising: (a) atleast one water soluble, randomly substituted partially N-, partialO-acetylated chitosan or derivative thereof of claim 1, (b) and at leastone buffer solution.
 3. The composition of claim 2, wherein the at leastone buffer solution comprises a borate buffer or a phosphate buffer. 4.The composition of claim 2, further comprising at least one biocidaladjuvant.
 5. The composition of claim 4, wherein the at least onebiocidal adjuvant comprises EDTA.
 6. The composition of claim 2, whereinthe pH of the composition is from 6 to
 8. 7. The composition of claim 2,further comprising at least one surfactant.
 8. A pharmaceuticalpreserving composition comprising the product formed from mixingcomponents a and b of claim
 2. 9. A contact lens solution comprising thepharmaceutical preserving composition of claim
 2. 10. A contact lenssolution comprising the product formed from mixing components a and b ofclaim
 2. 11. A method of preserving a contact lens solution, comprisingmixing a contact lens solution with the composition of claim
 2. 12. Themethod of claim 11, wherein components a and b are present in an amountsuch that the bacteria Staphylococcus aureus, Pseudomonas aeruginosa andEscherichia coli are reduced by at least 99.99% (3 logs) within 14 daysafter the challenge and re-challenge dates, each.
 13. The method ofclaim 11, wherein components a and b are present in an amount such thatthe growth of Aspergillus niger and Candida albicans is not allowedwithin 14 days after the challenge and re-challenge dates, each.
 14. Amethod of disinfecting a contact lens, comprising soaking the contactlens with the composition of claim 2 for a suitable period of time. 15.The method of claim 13, further comprising rubbing and rinsing thecontact lens with the composition of claim
 2. 16. A process forproducing a water soluble, randomly substituted partial N-, partialO-acetylated chitosan or derivative thereof, comprising the step ofreacting a randomly substituted partial N-, partial O-acetylatedchitosan or chitosan derivative with a base in a solvent.
 17. Theproduct produced by the method of claim
 16. 18. A phamaceuticalpreserving composition comprising: (a) at least one product of claim 17,(b) and at least one buffer solution.
 19. A pharmaceutical preservingcomposition comprising: (a) at least one chitosan or chitosanderivative, (b) at least one buffer solution.
 20. The composition ofclaim 19 wherein the at least one chitosan or chitosan derivativecomprises a chitosan salt, water soluble chitosan, randomly substituted,water soluble, partial N-, partial O-acetylated chitosan, chitosanoligosaccharide, carboxymethyl chitosan, or hydroxyalkyl chitosan. 21.The composition of claim 19, wherein the at least one chitosan orchitosan derivative comprises glycol chitosan, hydroxypropyl chitosan,dihydroxypropyl chitosan, hydroxybutyl chitosan or dihydroxybutylchitosan.
 22. The composition of claim 19, wherein the at least onebuffer solution comprises a borate buffer or a phosphate buffer.
 23. Thecomposition of claim 19, further comprising at least one biocidaladjuvant.
 24. The composition of claim 23, wherein the at least onebiocidal adjuvant comprises EDTA.
 25. The composition of claim 19,wherein the pH of the composition is from 6 to
 8. 26. The composition ofclaim 19, further comprising at least one surfactant.
 27. A contact lenssolution comprising the pharmaceutical preserving composition of claim19.
 28. A contact lens solution comprising the product formed frommixing components a and b of claim
 19. 29. A pharmaceutical preservingcomposition comprising the product formed from mixing components a and bof claim
 19. 30. A method of preserving a contact lens solution,comprising mixing a contact lens solution with the composition of claim19.
 31. The method of claim 30, wherein components a and b are presentin an amount such that the bacteria Staphylococcus aureus, Pseudomonasaeruginosa and Escherichia coli are reduced by at least 99.99% (3 logs)within 14 days after the challenge and re-challenge dates, each.
 32. Themethod of claim 30, wherein components a and b are present in an amountsuch that the growth of Aspergillus niger and Candida albicans is notallowed within 14 days after the challenge and re-challenge dates, each.33. A method of disinfecting a contact lens, comprising soaking thecontact lens with the composition of claim 19 for a suitable period oftime.
 34. The method of claim 33, further comprising rubbing and rinsingthe contact lens with the composition of claim
 19. 35. A process forproducing a water soluble, randomly substituted partial N-, partialO-acetylated chitosan or chitosan derivative, comprising the steps ofdissolving a chitosan or chitosan derivative in an aqueous acidicsolution and reacting the chitosan or chitosan derivative with anacetylating agent in the presence of a phase transfer reagent.
 36. Theprocess of claim 35, wherein the water soluble, randomly substitutedpartial N-, partial O-acetylated chitosan or derivative dissolves insolutions that have a near neutral pH value.
 37. The process of claim36, wherein the near neutral pH value is from pH 6.0 to pH 8.0.
 38. Theprocess of claim 35, wherein the phase transfer reagent is comprised ofat least one quaternary ammonium salt of Equation I:[A]w[B]x[C]y[D]zN+Q  Eq. I wherein: w, x, y and z are independentlyselected as integers from 0 to 4, with the sum of w, x, y, and z equalto 4; Q is a counter-ion selected from the group consisting of F⁻, Cl⁻,Br⁻, I⁻, CH₃COO⁻, OH⁻, HSO₄ ⁻, NO₃ ⁻, PF₆ ⁻, BF₄ ⁻, HCOO⁻ and H₂PO₄ ⁻;and A, B, C and D are independently selected from the group consistingof C₁-C₁₈ alkyl; phenyl in which the phenyl ring is unsubstituted orsubstituted by C₁-C₈ alkyl, C₁-C₈ alkoxy, halo, hydroxy, phenoxy, nitro,carboxy, acetamido, or aryl; benzyl; C₅-C₆ cycloalkyl; or heterocyclicring system.
 39. The process of claim 38, wherein the quaternaryammonium salt is benzyltriethylammonium chloride, tetrabutylammoniumbromide, tetramethylammonium chloride, tetrabutylammonium iodide,tetrabutylammonium dihydrogen phosphate, or a mixture thereof.
 40. Theprocess of claim 35, wherein the phase transfer reagent is comprised ofat least one quaternary phosphonium salt of Eq. II:[A]w[B]x[C]y[D]zP+Q⁻  Eq. II wherein: w, x, y and z are independentlyselected as integers from 0 to 4, with the sum of w, x, y, and z equalto 4; Q is a counter-ion selected from the group consisting of F⁻, Cl⁻,Br⁻, I⁻, CH₃COO⁻, OH⁻, HSO₄ ⁻, NO₃ ⁻, PF₆ ⁻, BF₄ ⁻, HCOO⁻ and H₂PO₄ ⁻;and A, B, C and D are independently selected from the group consistingof C₁-C₁₈ alkyl; phenyl in which the phenyl ring is unsubstituted orsubstituted by C₁-C₈ alkyl, C₁-C₈ alkoxy, halo, hydroxy, phenoxy, nitro,carboxy, acetamido, or aryl; benzyl; C₅-C₆ cycloalkyl; or heterocyclicring system.
 41. The process of claim 40, wherein the quaternaryphosphonium salt is hexadecyltributyl phosphonium bromide, tetrabutylphosphonium bromide, or a mixture thereof.
 42. The process of claim 35,wherein the phase transfer reagent comprises at least one crown ether.43. The process of claim 42, wherein the crown ether is 15-crown-5,18-crown-6, cis-dicyclohexano-18-crown-6, dibenzo-18-crown-6, or amixture thereof.
 44. The process of claim 35, wherein the phase transferreagent comprises at least one pyridinium salt.
 45. The process of claim44, wherein the pyridinium salt is 1-cetylpyridinium bromidemonohydrate, 1-dodecylpyridinium chloride mono-hydrate,1-benzyl-2-hydroxy pyridinium chloride, or a mixture thereof.
 46. Theprocess of claim 35, wherein the acetylating agent is acetic anhydride.47. The process of claim 35, further comprising isolating the watersoluble, chitosan or derivative thereof from the phase transfer reagent.48. The product produced by the method of claim
 35. 49. The productproduced by the method of claim
 38. 50. The product produced by themethod of claim
 39. 51. The product produced by the method of claim 40.52. The product produced by the method of claim
 41. 53. The productproduced by the method of claim
 42. 54. The product produced by themethod of claim
 43. 55. A pharmaceutical preservative compositioncomprising: (a) at least one product of claim 48, (b) and at least onebuffer solution.